Page 124 - Haematologica August 2018

J. Muller et al.
sible for side effects such as osteonecrosis of the jaw,
renal impairment, atypical fractures and hypocalcemia.5,6 These limitations highlight the need for new therapeutic strategies that ideally have a combined anti-MM and anti-MMBD effect.
We recently reported that maternal embryonic leucine zipper kinase (MELK) expression is strongly associated with proliferative high-risk MM, and that MELK inhibi- tion with a small molecule inhibitor, OTSSP167, reduces tumor load in a murine MM model.7 Overexpression of MELK as well as an inverse correlation between MELK expression and survival has been reported for multiple malignancies.8-10 MELK promotes cell cycle progression and interacts with M-phase inducer phosphatase 2 (CDC25B) and co-localizes with key cell cycle regulators such as cyclin B1 and cyclin-dependent kinase 1 (CDK1).11 Downstream targets of MELK include the tran- scription factor forkhead box protein M1 (FOXM1)12 and the histone-methyltransferase enhancer of zeste homolog 2 (EZH2).13 Of note, FOXM1 can also directly regulate MELK expression,10 presumably resulting in a positive feedback loop, and has been identified as a ther- apeutic target for high-risk MM.14
The role of MELK and FOXM1 in osteoclasts and osteoblasts has not yet been explored. Regarding EZH2, Fang et al. showed that EZH2 promotes osteoclastogene- sis by reducing the transcription factor IRF8 and subse- quent upregulation of NFATC1.15 In addition, EZH2 pre- vents osteogenic differentiation of mesenchymal cells and suppression of runt-related transcription factor 2 (RUNX2) has been implicated in this process.16 Inhibition of EZH2 has been shown to mitigate bone loss in ovariectomized mice17 and to reverse MM-induced sup- pression of osteoblast differentiation in vitro.18
Because the described roles of downstream targets of MELK suggest that its inhibition could concurrently block osteoclast function and stimulate osteoblast func- tion, we examined the potential of OTSSP167 as a novel therapeutic agent for MMBD.
Methods
Reagents
OTSSP167 (Biorbyt) was dissolved in DMSO and stored at - 20°C. For in vivo studies, OTSSP167 was dissolved in 0.5% methylcellulose (Sigma-Aldrich) and stored at -20°C. The fol- lowing antibodies were used: anti-FOXM1 (SC-502, Santa Cruz), anti-EZH2 (#4905, Cell Signaling Technology) anti-MELK (GTX111958, GeneTex and 2274S, Cell Signaling Technology), anti-a-tubulin (T6074, Sigma), anti-GAPDH (2118, Cell Signaling Technology), anti-rabbit-HRP (P0217, Agilent) and anti-mouse HRP (P0260, Agilent).
Cells and culture conditions
Human peripheral blood mononuclear cells (PBMCs) were obtained after Ficoll (GE Healthcare) separation of whole blood. RAW264.7 cells and 5TGM.1GFP+ cells were cultured in DMEM (Lonza) supplemented with 10% fetal bovine serum (FBS)(Sigma-Aldrich), 2mM L-glutamine (Lonza) and 1% peni- cillin/streptomycin (P/S) (Lonza). TERT+ bone marrow mes- enchymal stromal cells (BMSC-TERT) (kindly provided by Dr. D Campana, St. Jude Children’s Research Hospital, Memphis, TN, USA) were cultured in RPMI-1640 (Gibco) supplemented with 10% FCS, 2mM L-glutamine and 1% P/S.
Cell viability assay and cell cycle analysis
RAW264.7 and PBMC viability were assessed with the cell proliferation kit I (Roche). BMSC-TERT viability was assessed with the Cell Counting Kit 8 (Sigma-Aldrich). For cell cycle analysis, cells were stained using PI/RNase staining buffer (BD Biosciences), followed by FACS analysis on a FACSCalibur (BD Biosciences).
Osteoclast differentiation and in vitro bone matrix resorption
PBMCs were seeded at a density of 750,000 cells/cm2 in alpha- MEM (Lonza) supplemented with 10% FCS, 2 mM L-glutamine and 1% P/S. Cells were left to adhere for 4 hours. Next, the medi- um was refreshed and supplemented with 25 ng/ml human M- CSF and 50 ng/ml human sRANKL (Peprotech). The culture medi- um was refreshed twice per week and cultures were stopped on day 14. RAW264.7-derived osteoclast cultures were established as described previously.19 TRAP activity in osteoclast cultures was detected using the Leukocyte TRAP kit (Sigma-Aldrich). Alternatively, cultures were lysed for RNA or protein extraction. Bone resorption by osteoclasts was assessed in Osteo Assay 96- well plates (Corning) as described previously.19 Actin ring forma- tion was assessed by staining cultures with phalloidin-FITC (Sigma-Aldrich), followed by analysis on an A1R confocal fluores- cent microscope (Nikon).
Quantification of reactive oxygen species
Reactive oxygen species (ROS) were detected using the Cellular Reactive Oxygen Species Detection Assay kit (Abcam). In short, cells were stained with DCDFA for 30 minutes at 37 oC and fluo- rescence was measured (Ex/Em = 485/535 nm) on an Infinite M200 Pro plate reader (Tecan).
Osteoblast differentiation and functional analyses
BMSC-TERT were seeded at a density of 25,000 cells/cm2 and grown to 70–80 % confluence. Osteoblast differentiation was ini- tiated by changing the medium to alpha-MEM supplemented with 10 % FCS, 2 mM L-glutamine, 1% P/S, 100 nM dexametha- sone, 50 μg/ml ascorbic acid and 3 mM b-glycerophosphate (Sigma-Aldrich). Osteogenic medium was refreshed twice per week. Collagen secretion and matrix mineralization were assessed by Sirius Red and Alizarin Red staining, respectively, as described previously.19,20
Real-time PCR
Real-time PCR was performed as described previously19 using 250 nmol/L of the appropriate primers (IDT, Online Supplementary Table S1) or pre-designed Taqman gene expression assays (Applied Biosystems). Gene expression was normalized to b-actin and b2- microglobubulin expression (osteoclasts) or RPLP0 (osteoblasts). Measurements were performed at least in triplicate and relative expression levels were determined using the ΔCt method.
Western blotting
Cells were lysed in RIPA Lysis and Extraction buffer (Thermo Scientific) supplemented with cOmplete Protease Inhibitor Cocktail (Roche). Twenty μg of protein were separated by gel electrophoresis on a 10% SDS-polyacrylamide gel and transferred onto PVDF membranes (BioRad). Membranes were blocked with 5% BSA/PBS/Tween20 and incubated overnight at 4°C with pri- mary antibodies (MELK: 1:1000, FOXM1: 1:100, EZH2: 1:1000, a-tubulin: 1:5000, GAPDH: 1:2000). The next day, blots were incubated with a HRP-conjugated secondary antibody (1:5000), followed by visualization on an ImageQuant LAS4000 (GE Healthcare).
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